Over the past decade, it has been repeatedly demonstrated that homogeneity in electrochemical performance of lithium-ion cells plays a major role in determining the life and safety of lithium-ion battery modules or packs. Generally, the homogeneity of a battery pack is evaluated by characterizing the cells individually in terms of capacity, mass, impedance. Particularly, high quality electrochemical data heavily relies on the availability of high precision current source to minimize the discrepancy induced by the channel-to-channel variation. Here, a facile and precise measurement method is reported for screening cell-to-cell variations, in which voltage is the only indicator parameter independent of high precision current source. In detail, by connecting the cells in series (CiS), the measurement error of electrochemical data caused by stability and discrepancy of current sources among different charge/discharge equipment can be effectively avoided. The findings of this work showed that the cell-to-cell variations can be simply and sensitively detected with CiS configuration. For example, the relative standard deviation, which is the evaluation criterion of battery homogeneity, was 2.14% based on CiS while it was 0.43% based on individual measurements. The simple and precise CiS measurement is promising for evaluation of cell quality or module integration quality. In addition, this work can also provide a solid foundation for the development of detection algorithms for battery management systems to rapidly monitor battery homogeneity. However, a reliable and long-lasting power system is determined mostly by the cell homogeneity rather than the performance of individual cells9. The cell-to-cell variations in terms of capacity and impedance will subject individual cells to different levels of state of charge (SOC), current, temperature and aging, which in turn would accelerate the degradation of electrochemical performance or even lead to safety performance of the power system13. For instance, a weak cell with a smaller capacity than the average in a series-connected module will be repeatedly overcharged (over-discharged) during charging (discharging) process if the charge/discharge cutoff condition is determined according to the total voltage14. The weaker cell will then have faster decay than others and will be susceptible to failure under harsh conditions. The available energy of the module will also be limited by the weakest cell15. Therefore, there is an urgent need to understand and minimize the cell-to-cell variations in a battery module.Lithium ion batteries (LIBs) have to be integrated into modules and packs for large-scale applications such as electric vehicles (EVs) and stationary energy storage systems 11, including manufacturing factors , and environmental factors such as temperature gradient within the battery pack16. Rumpf et al.12 has evaluated the cell-to-cell variation due to manufacturing tolerances based on tests on 1100 commercial lithium-ion cells, and found relative variations of 0.28% and 0.72% for the cell capacity and impedance, respectively. Dubarry et al.17 has showed that cell-to-cell variations can drastically affect the performance and the reliability of both series and parallel battery packs. Baumhöfer et al.18 and Harris et al.19 showed that variation caused by production will result in a spread in battery degradation performance. Furthermore, theoretical analysis and industrial practice have showed that inhomogeneity of electrochemical characteristics in the unmatched cells inevitably resulted in voltage/SOC variations which can cause permanent internal damage in battery packs and energy storage systems20.There are different factors leading to variations among cells21. In order to achieve a stable configuration of a battery module or pack, a screening process is needed to ensure high homogeneity of the cells therein 23. Several screening processes have been previously proposed26. Multiple parameters, including capacity, mass, electrochemical impedance, direct current resistance and voltage, were employed to index the cell-to-cell variation. Rumpf et al.12 conducted a statistical analysis of parametric cell-to-cell variation of 15 different cell parameters and the correlations between parameters based on 1100 examined cells, and found it necessary to characterize variation in ohmic impedance when determining cell-to-cell variation. Dubarry et al.28 have conducted both statistical and electrochemical analyses to characterize cell-to-cell variations, and found that the cell capacity, resistance and rate capability can be regarded as three independent parameters for cell variations evaluation. Furthermore, as cell parameters show different evolution trends during aging, online algorithms are required to evaluate cell-to-cell variation during aging process15. Kum developed an overall cell selection framework with significant improvement in fabricating a module/pack with high homogeneity using second-life cells21. The proposed framework consisted of cell modeling, cell testing, parameter estimation technique, and a filtering algorithm. The screening result was effective, but multiple parameters were involved in this framework and the detection algorithm is complicated, making it challenging for online operation. Several approaches have also been proposed based on precise characterization of the electrochemical performance on individual cells. However, the measurement precision is a considerable technical barrier in these cell screening methods37. The major barrier for single cell approach is the difficulty in precisely measuring or estimating cell capacity, which involves the integration of measured current containing electronic noise over an extended period of charging or discharging. High precision equipment with a high-end current source is usually required for accurate cell-to-cell homogeneity evaluation38. An excellent implementation of this strategy is the development of high precision columbic efficiency (HPCE) measurement, which is only suitable for small scale deployment mainly due to its extremely high cost40. As a result, a facile, precise and less parameter involved cell-to-cell variation evaluation process would be ideal for estimation or fabrication of a reliable stationary or portable power system.The energy loss and SOC estimation error can be minimized by reducing the cell-to-cell variationTherefore, a facile and precise evaluation method named as “Connection in Series” or “CiS”, was proposed to evaluate the cell-to-cell variation in this work. In the CiS method, cells are evaluated in series connection, and only the cell voltages are measured for cell consistency evaluation. CiS was demonstrated to be effective for cell-to-cell variation analysis for commercial grade batteries. The simplicity, reliability and availability of CiS process for cell selection with high homogeneity was verified for vehicular application and for evaluation of cell homogeneity from different cell makers. The CiS-based methodology research as shown in this work can provide important knowledge for the development of algorithms for battery management systems (BMS) to evaluate the homogeneity of battery packs and to forecast potential failures on the fly.11. The most important concern is that cell-to-cell performance variation exists after assembling the cells in series or parallel into a module. This has triggered a major research effort for developing a low cost, effective, reliable method to evaluate the performance homogeneity of cells in a battery pack24. As the cell-to-cell variation in capacity, impedance, and other parameters will be intrinsically reflected by their SOC variations in CiS approach, the key focus of CiS approach has evolved into selecting the most representative SOC values for effective and reliable evaluation.The cell-to-cell performance variation in a battery pack is traditionally indexed by the capacity, mass, direct current resistance, impedance, etc.0.8Co0.15Al0.05O2(NCA) and graphite/LiCoO2(LCO) cells. It can be seen that a difference of 1% in practical capacity led to a significant difference in voltage at the end of discharge by 100 mV and 205 mV for NCA and LCO cells, respectively. A difference of 205 mV in voltage can be easily translated into 4.1% of practical range for typical battery measurement equipment rated as 5 V maximum range. Moreover, the variation in the instant voltage at the end of discharge is affected by both the capacity variation and resistance variation. As a result, the instant voltage at the end of discharge will be more sensitive for the detection of cell-to-cell variation than capacity and resistance for indexing the cell-to-cell variation. Furthermore, it is known that cell capacity is calculated by multiplying the discharge duration by the coulombic current, and strongly depends on the precision of the current passing through the cell. To eliminate the discrepancy resulting from the coulombic current measurement, a Connection-in-Series (CiS) approach was proposed here to precisely screen the cell-to-cell variation. In this method, the homogeneity of cells was effectively indexed by the voltage at the end of discharge, and additionally by the voltage at the end of charge and by the voltage after a period of rest after discharge24. In CiS configuration, the measurement was carried out with identical amount of charge delivered to the cells in series, and the voltage data were acquired almost at the same time. The screening index of cell-to-cell variation transformed from current capacities to voltages, achieving more precise and less expensive screening of cell homogeneity. Besides the significant advantage for high precision with low cost, the methodology research of CiS-based configuration can also provide seamless knowledge integration into the development of smart battery management system (BMS). This is because the CiS configuration has already been implemented into large battery packs, and the high precision voltage tracking for each cell is the base for the BMS.As is well known, the slopes of discharge curves are the most sensitive at the end of charging/discharging. For example, Fig. Q) and median voltage  of each cell were recorded as indexes for cell-to-cell variation. The results are shown in Fig. Vmax and Vmin) were 3.63 V and 3.57 V, respectively, with a difference  of 0.057 V. The maximum and minimum capacities (Qmax and Qmin) were 3.156 mAh and 3.053 mAh, respectively, which differed from each other by only 0.103 mAh . The relative standard deviation (δ) for Q was calculated to be 0.63%, and that for Vmid was 0.43%. They were all lower than 1%, indicating excellent cell homogeneity.For this test, 80 cells (NCR18650BE) purchased from Panasonic, recognized as the top battery maker with high quality, were evaluated to compare the difference between CiS and state-of-the-art. For state-of-the-art test, both capacity (Vc-end), the instant voltage at the end of discharge (Vd-end), and the voltage after the end of discharge for 30 min (Vd-30min). The results are shown in Fig. ∆Vc-end, ∆Vd-end and ∆Vd-30min were 0.015 V, 0.294 V and 0.142 V, respectively. The relative standard deviations (δ) of Vc-end, Vd-end and Vd-30min were 0.09%, 2.14% and 0.93%, respectively. The SOCs of all cells were the same (0%) at the very beginning, and all the cells were charged the same capacities in series, reaching around 100%SOC. Therefore, a very small relative standard deviation (δ=0.09%) of Vc-end was observed. The difference in Vc-end was mainly caused by the precision of the instrument, uneven self-discharge and impedance variation. The 15 mV difference indicates only 0.3% relative error, which is within the instrument error for 5 V range measurement (0.5%). δ for Vd-30min was 0.93%, right between δ = 0.43% for Vmid and δ = 2.14% for Vd-end. This result will be applied in the next section.For CiS test, these 80 cells were connected in series for charge/discharge, and experienced the same current, same duration and same capacity. The cell voltage was the only measured parameter, as shown in Fig. Vd-end and Vd-30min exhibited good correlations with cell capacity, with the correlation coefficients reaching 0.6635 and 0.6434, respectively. However, Vc-end showed weak correlation with cell capacity. Moreover, correlations between cell resistance and Vd-end, Vd-30min and Vc-end were also presented in Fig. Vd-end and Vd-60s by the discharge current. Vd-60s was the cell voltage after the end of discharge for 60 s. Vd-end and Vd-30min also showed strong correlations with cell resistance. Therefore, we can conclude that Vd-end and Vd-30min measured by the proposed CiS method do have good correlations with cell capacity and resistance, and thus can be applied to evaluate cell-to-cell variations. Moreover, we can find that ∆Vd-end and its δ were very large, being 294 mV and 2.14% respectively. Compared with state-of-the-art test results (δ = 0.63% for Q and δ = 0.43% for Vmid), Vd-end was more sensitive to the cell-to-cell variation for the same group of cells. This verified the effectiveness of CiS and validity of Vd-end as an ideal index for cell-to-cell variation.As shown in Fig. δ value for Vc-end increased slightly from 0.10% to 0.12%. δ for Vd-30min increased considerably from 0.07% to 0.23%, but was still very small. δ for Vd-end nearly doubled from 0.57% to 0.99%. Moreover, ∆Vd-end also increased from 85 mV to 163 mV, equivalent to the capacity difference increase from 0.13% to 0.26% when calculated by the principle shown in Fig. 41.Due to the high sensitivity of homogeneity evaluation by CiS, the proposed CiS method was used to evaluate the possible aging differences between high quality cells shelved for 2.5 years. For this test, 80 cells from Panasonic were initially evaluated in December of 2015  and present varied chemical/physical states in the following time points47, and kinetic factors are generally presented as impedances in battery. In this sense, the rate dependence of cell-to-cell variations will help to identify the influencing factors for battery kinetics introduced during manufacturing11. However, the cell-to-cell variation dependence on C-rate is unclear. On one hand, the subtle variation in impedance can be magnified in numerical value at high C-rate. On the other hand, high C-rate operation may cause temperature rise inside the battery, which is known to help decrease the battery impedance. That is, high C-rate may lead to impedance reduction in numerical value. Furthermore, high rate charge/discharge implies high measurement error for normal charge/discharge instrument, which affects the analysis of cell-to-cell variation. Here, the preferable C-rate for CiS was determined, and the possible application of CiS at different C-rates was also presented. CiS approach was employed to investigate the rate dependence of cell-to-cell variations. The evaluation of cells from different manufacturers was further discussed.Both thermodynamic and kinetic factors contribute to the cell-to-cell variationsδ. Then, cell-to-cell variation was further checked at 0.5 and 1 C-rate. The results are shown in Fig. Vd-end was the most sensitive parameter for cell-to-cell variation. The charge tests were stopped when any cell reached 4.2 V. It was found that each Vc-end was very close to 4.2 V, indicating that the uniformity was good for charging process. The 20 cells of any brand selected according to Vd-end can ensure high homogeneity of Vc-end, proving the effectiveness of CiS. It is known that high homogeneity of Vc-end is important to avoid overcharging in a module or pack configuration.The cells produced by Panasonic , LG Chem  and Lishen  were evaluated respectively. Firstly, 20 cells with good consistency were selected among 80 cells by CiS at 0.1 C-rate according to Vd-30min, cell homogeneity at different C-rates can be roughly determined by the fluctuation range of the statistical curves. Then, it was found that cells from Panasonic exhibited good homogeneity at both 0.5 and 1.0 C-rate, as shown in the middle of Fig. For Vd-end of cell No.6 was higher than that of the two adjacent cells at 1 C rate, but it was lower at 0.1 C rate. That might be induced by variations in cell rate capability. Cell No.6 had a smaller capacity than the two adjacent cells and thus showed a lower Vd-end at 0.1 C. Inversely, cell No.6 exhibited a larger capacity when C-rate increased to 1 C, indicating that cell No.6 might has better rate capability than the adjacent cells28. The cells from LG Chem and Lishen also showed similar behavior, as presented in Fig. 28.Interestingly, the sequence of cell number changed in homogeneity screening by different C-rates, according to voltage value. For example, in Fig. δ) is proposed as an appropriate index for homogeneity. In statistics, δ is also known as the coefficient of variation, a standardized measure of dispersion for a probability distribution49. It was observed that the evolution trend of δ for Vd-end was similar to that for Vd-30min, as shown in Fig. Vd-end is a suitable index for cell-to-cell variation.Quantitatively, relative standard deviation  was proposed to be an appropriate index for cell homogeneity in parameter processing. Low C-rate was preferred for CiS, as the cell homogeneity decreased with decrease in C-rate. Compared with the state-of-the-art evaluation method11, the proposed CiS approach can save equipment cost, manpower, analysis test and data processing. Investigations on the variation of cell homogeneity with storage duration showed that cell homogeneity became worse during aging even though the cells started from quite similar physical and chemical states. Besides, the dependence of cell homogeneity on C-rate can be used as an index for quality control or quality evaluation. The information may help battery makers to improve cell design, manufacturing and quality control. CiS was shown to be a facile and highly precise approach for evaluating and tracking the consistency of lithium-ion batteries. It can not only be developed as a universal standard test protocol of cell-to-cell variation for vehicular and stationary energy storage application, but also have possible applications in cell screening.Cell homogeneity is critical for performance and durability of energy storage systems. The state-of-the-art evaluation of cell homogeneity based on individual cell performance involves intertwined parameters (such as capacity and impedance) and a large number of measurements. Based on the charge/discharge characteristics of lithium-ion batteries, a CiS method that indexes cell-to-cell variations only by a state voltage was proposed in this paper. In detail, the voltage at the end of discharge 0.8Co0.15Al0.05O2(NCA) for Panasonic cell, and was LiCoO2 (LCO) for LG Chem and Lishen cells. The anode material of the three kinds of cells was artificial graphite. The separators were all polypropylene. Neware test system was used to perform charge-discharge tests, and Arbin EVTS battery testing system was used to record cell voltage during charge-discharge.Cylinder cells (18650 type) were purchased from market and were not balanced before. The cell manufacturers were Panasonic , LG Chem  and Lishen , respectively. The cathode material was LiNiThe capacities and median voltages of Panasonic cells were measured individually before the CiS tests at 25 °C according to the product specification. The charge protocol was CC-CV with 1/5 C reaching an upper voltage limit of 4.2 V, and the cutting-off current was 0.05 C. After a rest period of 30 min, the cells were discharged with a constant current 1/3 C to 2.5 V.X C represents the current that can fully discharge the cell from 100%SOC to 0%SOC in 1/X hour. 1 C was 3.0 A for Panasonic NCR18650BE cells (3.0Ah), and 2.6 A for both LG Chem 18650B4 cells (2.6Ah) and Lishen LR1865SK cells (2.6Ah). Three instant voltages, i.e. the end of charging (Vc-end), discharging (Vd-end) and discharging for 30 min (Vd-30min), in the process were recorded and their discrete factors were calculated as the criteria for homogeneity evaluation.All cells with 100%SOC were connected in series. The voltage of every single cell was recorded by Arbin EVTS battery testing system (accuracy of voltage control was ±0.1% FSR). The homogeneity of 80 Panasonic cells at 2.5 year intervals was investigated by CiS. As shown in Fig. all cells were charged to 100% SOC. (the charge protocol refers to the battery specifications provided by manufacturer: charging to 4.2 V with 0.3 C constant current (CC) and then constant voltage (CV) charging with cut-off current of 0.05 C);the 20 cells were connected in series to form a module, and the voltages of individual cells were recorded;the module was discharged at 0.1 C to 3.0 V of any single cell;rest for 30 min;the module was then charged at 0.1 C up to 4.2 V of any single cell; then steps 3, 4, and 5 were successively repeated at 0.5 C and 1.0 C, respectively.To investigate the dependence of consistency to rate, 20 cells with the best homogeneity were selected among 80 cells for the three kinds of cells respectively. In this process, the charge/discharge rate was 0.1 C. Then the consistency of each kind of 20 cells was measured using CiS method at 0.5 and 1 C-rate for both charge and discharge at 25 °C. The test protocol was as follows:


The qualification run met the acceptance criteria of ±25% accuracy and precision values for quality control (QC) samples. The overall recovery was 42.61%. The dilution integrity was for a series of 5-fold dilutions with accuracy and precision values ranging within ±25%. The stability results indicated that acDrug was stable at all stability test conditions : 3 cycles). This qualified method was successfully applied to in vitro linker stability and pharmacokinetic case studies of acDrug in rats.The novel prenyl transferase-mediated, site-specific, antibody–drug conjugate LCB14-0110 is comprised of a proprietary beta-glucuronide linker and a payload  attached to human epidermal growth factor receptor 2 (HER2)-targeting trastuzumab. A LC-MS/MS method was developed to quantify the antibody-conjugated drug (acDrug) for in vitro linker stability and preclinical pharmacokinetic studies. The method consisted of affinity capture, enzymatic cleavage of acDrug, and LC-MS/MS analysis in the positive ion mode. A quadratic regression (weighted 1/concentration The antibody–drug conjugate (ADC) technology platform uses an antibody, linkers, and chemical drugs. Chemical drugs are conjugated to an antibody by chemical linkers to deliver the chemical drugs to the target . Target-In this paper, an affinity capture LC-MS/MS method for the novel prenyl transferase-mediated, site-specific, antibody-drug conjugate LCB14-0110 in rat plasma was developed for preclinical pharmacokinetic assessment. Beta-glucuronidase was used for specific cleavage of the novel prenyl transferase-mediated, site-specific conjugation, and the propriety beta-glucuronide linker attached to the antibody (trastuzumab) and the released chemical drug (MMAF) were analyzed by LC-MS/MS. This method was able to directly measure the chemical drugs linked to the antibody. The method was qualified and applied to an in vitro linker stability study and a preclinical PK study. To the authors’ best knowledge, this is the first assay for the quantification of acDrug for the novel prenyl transferase-mediated, site-specific antibody-drug conjugate using LC-MS/MS.3OH. The parent [M + H]+ ion was detected at m/z 732.5. The most abundant product ion was at m/z 700.5.LC-MS/MS, in the positive ion mode, was used for the quantification of acDrug . Method qualification was carried out with a ‘fit-for-purpose’ approach. Calibration curves with seven points in duplicate were freshly prepared for all data sets. The lower limit of quantification (LLOQ) of the assay was determined to be 19.17 ng/mL. The calibration curve range was 19.17–958.67 ng/mL. Representative chromatograms of the LLOQ (19.17 ng/mL) and high QC (479.33 ng/mL) are also shown in 2), with the equation y = ax2 + bx + c, was used to fit calibration curves over the concentration range of 19.17–958.67 ng/mL for acDrug. The coefficient of determination (r) value for calibration curves of acDrug was used to evaluate the fit of the curves. The correlation coefficients of the calibration curve were greater than 0.993 for acDrug. A quadratic regression (weighted 1/concentrationAccuracy (%) and precision (% coefficient of variation (CV)) of QC samples were evaluated for examining assay performance and the results are shown in The qualification run met the acceptance criteria of ±25% accuracy and precision for QC samples, which is acceptable for the early-stage drug discovery study.This quantification assay of acDrug consisted of four steps: (a) affinity capture of LCB14-0110, (b) cleavage of acDrug by beta-glucuronidase, (c) post-cleavage, and (d) LC-MS/MS analysis for the cleaved MMAF. In the recovery sample A (Ra), LCB14-0110 was added before affinity capture by magnetic beads. This sample was prepared by the standard protocol described in the As a result, the bottle neck of sample preparation step appeared to be the affinity capture step using magnetic beads. The total recovery could be improved if the number of magnetic beads was increased or more selective beads were used. The accuracy (%) and precision (%CV) of the dilution QC sample (5-fold dilution) met the acceptance criteria of ±25% . This meThe results for stability tests are summarized in The accuracy (%) and precision (%CV) of stability samples met the acceptance criteria of ±25%. As a result, acDrug in rat plasma was stable under the different experimental conditions .The qualified LC-MS/MS method was applied for an in vitro linker stability study and a preclinical PK study in rats. The in vitro linker stability samples were incubated in 37 °C for 0, 1, 3, 5, and 7 days. The result is shown in About 85.38% of acDrug remained on day seven in rat plasma. Only a small amount of MMAF from LCB14-0110 was released as free payload or catabolized in rat plasma. It was suggested that this ADC was very stable in plasma and no/little payloads would be released in this experimental condition.In vivo rat plasma samples obtained after intravenous administration of 3 mg/kg of LCB14-0110 were also analyzed by using this method. For acceptance of study sample analytical runs, at least two-thirds of the QC samples had to be within ±25% accuracy with at least half of the QC samples at each concentration meeting these criteria. The pharmacokinetic profile of acDrug is shown in n = 3, total antibody (tAb) and free payload also had to be analyzed through the identical bioanalytical plasma sample obtained from each individual group, so the remaining plasma sample was pooled for the analysis of acDrug. Therefore, the standard deviation value was not expressed. Since the standard deviation from the analytical results of tAb and free payload showed very low values (data not shown), we anticipated that the standard deviation of acDrug would also be very low.From the PK perspective, the half-life of acDrug from LCB14-0110 was similar to that of trastuzumab. It means that the linker of LCB14-0110 is very stable in systemic circulation in vivo. These results also correlated well with the in vitro linker stability study. The qualified LC-MS/MS method was thus successfully applied to an in vitro linker stability study and a preclinical PK study in rats.LCB14-0110, a HER2-targeting ADC based on trastuzumab , was obtVerapamil and beta-glucuronidase were purchased from Sigma-Aldrich . Formic acid was purchased from Daejung Chemical . All other chemicals were commercial products of analytical or reagent grade and used without further purification.LCB14-0110 stock solutions were prepared in phosphate-buffered solution (PBS) and stored at −80 °C. The calibration curve was made by seven calibration standards at the final plasma concentration range of 19.17~958.67 ng/mL. Final plasma concentrations of QC samples were 239.67 (low QC) and 479.33 (high QC) ng/mL. Verapamil was used as the internal standard (IS).Each 50 μL aliquot of study samples, QCs, and STDs were mixed with 374 μL of PBS and 30 μL of magnetic bead suspension. After gently shaking at room temperature for 2 h, the magnetic beads were washed using 600 μL of PBS, two times. Forty microliter of 1 mg/mL beta-glucuronidase in PBS was added to tubes containing the washed magnetic beads to release MMAF from LCB14-0110. After shaking the mixtures for 1 min, the mixtures were incubated for 3 h at 37 °C. Forty microliter of acetonitrile containing IS was added to the mixtures to quench the activity of beta-glucuronidase. After shaking the mixtures for 1 min, the mixtures were centrifuged at 7000 rpm for 7 min and 60 μL of supernatant was mixed with 60 μL of water for LC-MS/MS analysis.TM 5600 mass spectrometer . The HPLC analytical column used was a Kinetex XB-C18 column . The mobile phase consisted of: mobile phase A, distilled and deionized water containing 0.1% formic acid; and mobile phase B, acetonitrile containing 0.1% formic acid. The gradient was as follows: from 0 min to 0.5 min, 5% B; from 0.5 min to 1.7 min by a linear gradient from 5% B to 95% B; 95% B was maintained for 0.3 min; from 2.0 min to 2.1 min by a linear gradient from 95% B to 5% B; and then 5% B was maintained for 1.4 min for column re-equilibrium. The gradient was delivered at a flow rate of 0.4 mL/min and the injection volume was 10 μL.The liquid chromatography–high-resolution mass spectrometric system consisted of a Shimadzu CBM-20A HPLC pump controller , two Shimadzu LC-20AD pumps, a CTC HTS PAL autosampler , and a quadrupole time-of-flight TripleTOFm/z 100~800 for both TOF-MS scan and product ion scan. For the quantification, [M + H]+ ion of MMAF and IS  were selected and their product ions at m/z 700.5 and 165.1 were used for quantitative analysis, respectively. The source temperature was set at 500 °C with a curtain gas flow of 30 L/min. The ion spray voltage was set at 5500 V. For MMAF and IS, declustering potential was 100 V and 100 V, and the collision energy was 36 V and 30 V, respectively.The TOF-MS scan mass spectra and the product ion scan mass spectra were recorded in the positive ion mode. The scan range was 2 + bx + c after 1/concentration2 weighting. In addition, two blank plasma samples were run. The accuracy and precision were calculated at each QC concentration.Method qualification was carried out with a ‘fit-for-purpose’ approach. The qualification run contained duplicate standards at seven concentrations and QCs at two concentrations. The acceptance criteria for standards and QCs in the qualification run were within ±25% of the precision and accuracy values. Calibration was done by establishing a quadratic regression function with the equation y = axOverall, recovery from the affinity capture, cleavage of beta-glucuronidase and the post-cleavage process of LCB14-0110 was also assessed. The dilution integrity test was conducted by 5 times dilution using rat plasma. The acceptance criteria for dilution integrity were within ± 25% of the precision and accuracy values. Stability tests were conducted in rat plasma sample under different conditions, such as short-term, long-term, and freeze/thaw stability at low and high QC. The short-term stability was determined at room temperature for 1 day. The long-term stability was determined by analyzing QC samples kept frozen at −80 °C for 10 months. For the freeze/thaw stability, the samples were subjected to three freeze-and-thaw cycles at −80 °C. The acceptance criteria for all stability tests were within ±25% of the precision and accuracy values.® version 8.0.0 .LCB14-0110 in rat plasma was used for an in vitro stability study. The samples were incubated at 37 °C for 0, 1, 3, 5, and 7 days. After incubation, the samples were stored at −80 °C before analysis. The preclinical PK study was conducted in Sprague−Dawley rats. Animal experiments followed the animal care protocol (no. CNU-00685) approved by the Chungnam National University. All procedures related to animal experiments were also performed in accordance with the guidelines established by the Association for Assessment and Accreditation of Laboratory Animal Care International . LCB14-0110 was administered to rats via single intravenous bolus injection (3 mg/kg). The blood sampling times were 0, 0.002, 0.042, 0.125, 0.25, 1, 2, 3, 4, 7, 9, 14, 17, 21, and 28 days and each of the blood samples was collected in heparinated tubes. Blood samples were centrifuged and the supernatant plasma was stored at −80 °C. Pharmacokinetic calculations were performed using WinNonlinLCB14-0110 is a novel antibody–drug conjugate comprised of MMAF attached to HER2-targeting trastuzumab through a novel site-specific conjugation. This novel site conjugation method is comprised of the insertion of the CaaX sequence at the C-terminal of the protein binder, prenylation using farnesyltransferase, and drug conjugation through an oxime ligation reaction . MMAF an2 weighting, which was sufficient enough to cover the pharmacokinetics of LCB14-0110 acDrug in rats when administered at 3 mg/kg IV. This affinity capture LC-MS/MS method was accurate and reproducible for the determination of acDrug concentration. LCB14-0110 showed excellent PK profile with enhanced linker stability, which could potentially improve the efficacy and safety in clinical studies. The results in this paper will help studies related to ADCs with a similar linker platform. The limitation in this paper is that the affinity capture LC-MS/MS method measured the parent form of the antibody-conjugated payload, but not other catabolites simultaneously. If other catabolites were also active with a pharmacological activity as well as the parent payload, the quantitation of catabolites as well as the parent payload simultaneously would be helpful to understand the pharmacological effect of the ADC.In this paper, an affinity-capture LC-MS/MS method for LCB14-0110 in rat plasma was successfully developed and applied for in vitro linker stability and preclinical pharmacokinetic assessment in rats. Beta-glucuronidase used in this study was able to specifically cleave the novel prenyl transferase-mediated, site-specific conjugation and the propriety beta-glucuronide linker attached to the antibody. The method was qualified in a dynamic range of 19.17~958.67 ng/mL using quadratic regression with 1/concentration


The placenta provides nutrients and oxygen to embryo and removes waste products from embryo’s blood. As far as we know, the effects of exposure to Wi-Fi (2.4 GHz) signals on placenta have not been evaluated. Hence, we examined the effect of prenatal exposure to Wi-Fi signals on anti-oxidant capacity, expressions of CDKNA1, and GADD45a as well as apoptosis in placenta and pregnancy outcome. Pregnant mice were exposed to Wi-Fi signal (2.4 GHz) for 2 and 4 hr. Placenta tissues were examined to measure the MDA and SOD levels. To measure SOD, CDKNA1, GADD45a, Bax, and Bcl-2 expressions were compared by real-time PCR analysis. TUNEL assay was used to assess apoptosis in placenta tissues. The results were analyzed by one-way analysis of variance (ANOVA) using Prism version 6.0 software. P-value< 0.05). Also, quantitative PCR experiment showed that SOD mRNA expression significantly increased in Wi-Fi signal groups. The data showed that CDKN1A and GADD45a genes were increased in Wi-Fi groups . The quantitative PCR and the TUNEL assay showed that apoptosis increased in Wi-Fi groups . MDA and SOD levels had significantly increased in exposed Wi-Fi signal groups , apoptosis and CDKN1A and GADD45a overexpression in mice placenta tissue. However, further experimental studies are warranted to investigate other genes and aspects of pregnancy to determine the role of Wi-Fi radiation on fertility and pregnancy.  Reproduction is an important function of a living creature, which involves both female and male genital organs. Recently, evidence showed that both non-ionizing and ionizing radiations induce epigenetic changes, genomic instability and oxidative stress in reproductive tissues , 2. RadiExposure to Wi-Fi signal might lead to absorption of some transmitted energy by cells. Moreover, concerns regarding exposure to radiofrequency electromagnetic field (RF-EMF) during perinatal life are elevated. Several reports showed that chronic and acute exposure to EMR can lead to morphological modification in tissues and biological cells , 6. It iCyclin-dependent kinase interacting protein 1 (CDKNA1) and growth arrest and DNA damage inducible alpha (GADD45a) proteins play an important role in key cellular processes. The CDKNA1 and GADD45a proteins interact for the regulation of several cellular functions, including genotoxic stress, cell cycle control, senescence, and DNA damage repair. These proteins cause to facilitate cells to enforce cell cycle arrest in promoting DNA repair  , 11. To the best of our knowledge, the effects of exposure to Wi-Fi signals on placenta has not been assessed. Hence in the present study, we examined the effect of prenatal exposure to Wi-Fi signals on oxidative stress, apoptosis, and expressions of DNA repair genes including (CDK1A-GADD45a) in pregnancy outcome and placenta in a mice model.Study design and populationThis study was designed as a prospective trial, conducted to examine the effects of prenatal (in uterus) exposure to Wi-Fi signals on the expressions of CDKNA1, and GADD45a in placenta and pregnancy outcome in an animal model. The institutional animal ethics committee of Shiraz University of Medical Sciences, which follows the National Institutes of Health guidelines for the care and use of animals, approved the protocols and experiments in this study. Pathogen-free 3-month-old plug-positive mice were enrolled in the study. Exclusion criteria included females with plug-negative after 4 days of mating and plug-positive females with unhealthy conditions during pregnancy.We prepared pathogen free 3-month-old mice from the animal laboratory of Shiraz University of Medical Sciences. Male and female mice were mated over a 4-day period. Female mice were screened daily, and plug-positive females were separated from males. Females that were still negative after 4 days of mating were not included. Fifteen plug-positive females were assigned to 5 groups. Pregnant mice in case group were exposed to Wi-Fi signal-associated electromagnetic fields (2.4 GHz frequency band) by a D-Link Wi-Fi router , starting 5 days after mating and ending 1 day before the expected delivery, and exposure protocol were grouped II-V . During the exposure time, data was exchanged between a laptop and the modem at a distance of 5 meters (placed in another room). The Wi-Fi exposure source operated on power level of 1 W and the Specific Absorption Rate (SAR) at the distance of 30 cm in animals’ head level was 0.09 W/kg. The SAR comes from the following formula:md are the tissue conductivity, electric field and tissue density, respectively   using a homogenizer  and centrifuged for 45 min (13000 rpm at 4 °C). Then, the supernatant was used to measure antioxidant activity.Malondialdehyde assessment To measure malondialdehyde (MDA) level, 100 µl of supernatant, 5 µl of butilated toluene hydroxide, and 400 µl of %5 triclorostic acid were mixed, and the prepared solution was centrifuged for 10 min at 300 rpm. After removing 200 µl of the obtained solution, 150 µl of thiobarbitoric was added, and the mixture was placed in 95 ºC oven for 1 hr . Then, it was preserved at -4 °C and the absorption rate was measured at 532 nanometer wavelengths . To deteSuperoxide dismutase assessmentSuperoxide dismutase (SOD) activity was calculated by the procedure described by Madesh and Balasubramanian. It is a colorimetric assay involving the generation of superoxide by pyrogallol autooxidation and inhibition of superoxide-dependent reduction of the tetrazolium dye MTT  to its formazan, which is measured at 570 nm . The color evolved was stable for many hours and was expressed as SOD units (one unit of SOD activity was defined as the amount of enzyme causing 50% inhibition in the MTT reduction rate) . Quantitative reverse transcription-PCRAll placenta specimens were obtained through vacuum aspiration. Each specimen was flash-frozen in liquid nitrogen and used for quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) analysis as described below.TM spectrophotometer , and then stored at -80 °C until cDNA synthesis. The cDNA was then synthesized using 1000 ng of total RNA in a first-strand complementary DNA synthesis reaction by the help of RevertAid™ First Strand cDNA Synthesis kit .SOD, CDKN1A, GADD45a, Bax and Bcl-2 genes expression were determined by qRT-PCR analysis using the β-actin gene as a reliable internal control. Total cellular RNA was isolated from tissue using RNA extraction kit . The quantity and quality of the obtained RNA were checked by measuring the ratio of optical density (OD) of 260/280 nm using Nanodrop-∆∆CT) method was used to compare mRNA levels of the test group versus control, which represents fold change in data analysis.Quantitative real-time PCR was performed using the ABI Biosystem step one and the RealQ Plus 2x Master Mix Green . In each reaction, 200 nM of each primer  was addeTUNEL assays Three slides from each placenta were evaluated for cells apoptosis detection using TUNEL assay kit  according to the manufacturer’s instructions. Briefly, the placenta tissue slices were deparaffinised in xylenes and rehydrated at gradient concentration of ethanol. Proteinase k was added to increase membrane permeability. TUNEL MIX, and PI (Propidium iodide) were used for labeling and tracing. After washing, the slices were examined with a light microscope  at 400x magnification. The numbers of total and TUNEL-positive cells were counted in each field. The results were expressed as the apoptotic index  ×100%.Statistical analysispost hoc: Tukey) using GraphPad Prism software, version 6.0  to compare control group. The P-value less than 0.05 was considered to be statistically significant.All data in this study are analyzed by variance using the statistical program. All the data are presented as mean±SEM of at least three independent experiments and analyzed by one-way analysis of variance (ANOVA) . Two groups of II and III (2 hr- 30 cm and 2 hr- 60 cm) showed no significant difference with the control group . Total SOD activity was also elevated in the group V (4 hr- 60 cm group) (4.24 U/mg tissue) versus the control group . However, the activity of SOD was higher in the group III (2hr-60cm) (2.23 U/mg tissue) as compared to the control group . In SOD activity, there were no significant differences between the group II (2 hr- 30 cm) and the control group   compared to the control group  .P-value<0.05). The significance level of all groups (II-V) was less than 0.05 compared to the control group. In addition, the relative fold changes in GADD45a expression in the intervention groups were significantly higher than that of observed in the control group. Despite variations in different exposure groups, the maximum elevation for both genes was observed in mice exposed to Wi-Fi radiation in the 4 hr- 30 cm protocol. The DNA repair genes (including CDK1A-GADD45a) in the placenta were also measured using the real-time quantitative PCR experiment. Overall, compared to the control group, there was an overall increase in the studied genes in the intervention groups. Real-time quantitative PCR showed that CDKN1A expression in all intervention groups was significantly different as compared to control group . In accordance with real-time quantitative PCR results, the highest levels of Bax gene expression were found in group IV, 4 hr- 30 cm followed by group V, 4 hr- 60 cm , compared to the control group . There was no significant difference between the expression of Bax and bcl-2 in the 2 hr groups compared to the control group. Bax/Bcl-2 ratio is a measurable aspect of the apoptosis progression, which seems to determine the fate of apoptosis. The Bax/Bcl-2 ratio was increased in group V, 4 hr- 60 cm (6.86 fold). These data suggested that Wi-Fi radiation in the group V)4 hr- 60 cm) serves as an inducer of apoptosis through the activation of apoptotic genes as shown in P-value =0.0002). We assumed that Wi-Fi radiation might induce apoptosis in the placenta by increasing the expression of Bax and reducing the expression of Bcl-2. The expression analysis results indicated that exposure to Wi-Fi radiation , significantly down-regulated the Bcl-2 (0.45 fold), compared to the control group  on differentiation and proliferation of spermatogonia to be correlated with serum sex hormone levels. In parallel with a defect in spermatogenesis process, negative effects of Wi-Fi (2.4 GHz) on apoptosis status and histopathological changes of rat testis are possible . HoweverROS are produced continuously by the placenta, and they are neutralized by antioxidants present in the tissue. Also, in order to study the role of oxidative stress in placental dysfunction, sensitive methods for the detection of oxidative stress are necessary. Oxidative stress is a critical factor in many problems during pregnancy; thus, uterine problems in pregnancy could result in an imbalance of antioxidant/oxidant activity when antioxidant capacity cannot keep pace with increased oxygen tension, leading to a chronic state of oxidative stress .in vitro (Previous experiments on the effects of extremely-low-frequency (ELF)-EMF on preimplantation embryos also hypothesized that the adverse effects of ELF-EMF on preimplantation embryos might be caused by the DNA damage in the embryos in vitro . Howeverin vitro . ConsequOptimal fetal growth depends on functioning maternal, placental and fetal factors, the external environment, in combination with genetically predetermined growth potential. Fetal growth restriction (FGR) might occur as a result of defective function . Human pin vivo for animals, because apoptotic cells are eliminated quite rapidly. The importance of these findings lies in the role of apoptosis, which is associated with problems in placenta and embryo  to a CDMA (code division multiple access) signals or simultaneously to CDMA and WCDMA (wideband- CDMA) signals throughout the entire gestation period  are increased following treatment with DNA-damaging agents and stressful growth arrest conditions. These proteins play a critical role in the regulation of DNA repair, cell growth, and apoptosis, in response to stressful conditions . The resal cells . Furthernd Bcl-2 . Our datIn summary, our results provide evidence that Wi-Fi signals increase oxidative stress in placenta tissue. It was suggested that CDKN1A and GADD45a overexpression in the placenta tissue was caused by exposure to Wi-Fi radiation, and increase in apoptosis-positive cells and Bax/Bcl-2 ratio in the placenta tissue of mice were observed, especially in 4 hr- 30 cm group. However, further experimental studies are warranted to investigate other genes as well as other aspects of pregnancy to determine the role of Wi-Fi radiation on fertility and pregnancy.


We aimed to investigate the expressions of miR-21 and miR-210 in the breast cancer tissue and their correlation with clinicopathological features and prognosis.A retrospective analysis was performed on 68 patients with breast cancer treated surgically in Wuhan General Hospital of Guangzhou Military in 2014–2015. The breast cancer tissue and the adjacent normal tissue were collected from the patients. Quantitative real-time PCR (qRT-PCR) was used to detect the expression levels of miR-21 and miR-210 in the breast cancer and adjacent normal tissues.P<0.05), which were significantly correlated with lymph node metastasis, clinical staging and differentiation of patients (P<0.05). miR-21 and miR-210 were significantly positive correlated in both breast cancer tissues and adjacent normal tissues . The survival rate in the miR-210 high expression group was significantly lower than that in the miR-210 low expression group (P<0.05), whereas there was no significant difference between the miR-21 high and low expression groups.According to qRT-PCR, the expression levels of miR-210 and miR-21 in the breast cancer tissue were significantly higher than those in the adjacent normal tissue (miR-21 and miR-210 are highly expressed in the breast cancer tissue and significantly correlated with lymph node metastasis, clinical staging and differentiation. miR-210, the up-regulated expression of which is related to the poor prognosis of patients with breast cancer, may be a potential prognostic indicator for breast cancer, which can be used to judge the prognosis. As a malignant tumor in women with the highest incidence rate, breast cancer accounts for one quarter of female cancers, with a mortality rate accounting for 15% . The proAs a miRNA used for diagnostic research, miR-21 functions as a proto-oncogene , and is There are currently few reports on miR-21 and miR-210 in breast cancer, and there is no research report on whether there is significant correlation between miR-21 and miR-210. Therefore, miR-21 and miR-210 in the breast cancer tissue were explored in this paper in terms of their expression levels, their correlation with clinicopathological features, the correlation between the two and their effects on the prognosis.A total of 68 patients with breast cancer treated surgically in Wuhan General Hospital of Guangzhou Military, China from January 2014 to May 2015 were enrolled in this study, aged 30–67 yr old with an average age of 42.77±8.62 yr old. The breast cancer and adjacent normal tissues from the patients were obtained for experiments. There were 22 cases in clinical stage I, 21 cases in stage II, and 25 cases in stage III. General information is shown in The breast cancer tissue and the normal tissue without cancer cell infiltration under the microscope which was over 2 cm from the tumor margin were immediately taken after operation and stored in a refrigerator at −80 °C. All patients cooperated with medical staff to complete relevant diagnosis and treatment. Patients and their families were informed in advance before the study and signed an informed consent form.Reverse transcription kit Fermentas was purchased from Beijing Think-Far Technology Co., Ltd., qRT-PCR kit from Thermo Fisher Scientific, China, Trizol kit from Shanghai Pufei Biotech Co., Ltd., UV-3100PC spectrophotometer from Shanghai Mapada Instruments Co., Ltd.The total RNA in the breast cancer and adjacent normal tissues was extracted with one-step extraction method using the Trizol kit, with specific steps carried out in strict accordance with the instructions. The UV-3100PC spectrophotometer was used to detect the concentration and purity, 1% denatured agarose gel electrophoresis to detect the integrity. The RNA extracted was reversely transcribed into cDNA as a template for experiments. Primer sequences were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd., and U6 was used as an internal reference gene in this experiment. The reverse transcription system was 15 μL in total: 0.5 μL of primers for reverse transcription, 0.5 μL of reverse transcriptase, 1.5 μL of buffer, 2 μL of RNA, DEPC-treated water used to complement to 15 μL. Reaction conditions were at 37 °C for 10 min and at 95 °C for 5 min. The synthesized cDNA was stored at 4 °C.–ΔCT was used to analyze the expression levels of miR-21 and miR-210. The primer sequences are shown in The quantitative real-time PCR (qRT-PCR) reaction system was prepared based on the instructions. The system was 20 μL in total: 10 μL of 2×TaqPCR Master Mix, 1 μL of primers, 1.33 μL of cDNA (diluted at 1: 15), 10 μL of TaqMan 2× Universal PCR Master Mix II, DEPC-treated water used to complement to 20 μL. qRT-PCR instrument was used for PCR amplification, and reaction conditions were as follows: predenaturation at 95 °C for 10 min, at 95 °C for 15 s, at 65 °C for 30 s and at 72 °C for 30 s, for 40 cycles. PCR products were stored at 4 °C. There were 3 same samples in each group, and 2The relationship between miR-21 and miR-210 in breast cancer patients and adjacent normal tissues was observed, and the relationship between miR-21 and miR-210 and clinicopathological features were explored. The patients were followed up for three years for their survival situation. The relationship between miR-21, miR-210 expression and patient survival rate were analyzed; the correlation between miR-21 and miR-210 in adjacent tissues and cancer tissues was analyzed.P <0.05 indicates a statistically significant difference.SPSS20.0 software package  was used to statistically analyze the data, GraphPad Prism 7 to plot figures. Measurement data were expressed as mean±standard deviation, and t test was used for comparison between the two groups. Count data were tested by chi-square. Pearson correlation coefficient was used for data confirming to bivariate normal distribution, Kaplan-Meier for survival analysis, Log-rank for test. t= 2.921, P=0.004). The expression of miR-210 in the breast cancer tissue was (1.16±0.46), significantly higher than (0.85±0.27) in the adjacent normal tissue  (The expression of miR-21 in the breast cancer tissue was (4.77±1.36), significantly higher than (4.17±1.01) in the adjacent normal tissue (P<0.001) .P<0.05)  .P<0.05)  .P <0.001)  –2.P<0.05).The survival data of the breast cancer patients were counted, and the average value (1.04) of miR-210 expression was used as a boundary. Of 68 cases, 31 with a miR-210 value less than 1.04 were in the low expression group, another 37 with a miR-210 value greater than or equal to 1.04 in the high expression group. The survival rate was 48.65% in the high expression group and 83.87% in the low expression group, with follow-up time up to June 1, 2018. As shown in The survival data of breast cancer patients were counted, and the average value (4.24) of miR-21 expression was used as a boundary. Of 68 cases, 33 with a miR-21 value less than 4.24 were in the low expression group, another 35 with a miR-21 value greater than or equal to 4.24 in the high expression group. The survival rate was 60.00% in the high expression group and 69.70% in the low expression group, with follow-up time up to June 1, 2018. As shown in Breast cancer is a common malignant tumor in women , and itsBreast cancer is more common in people aged 40–60 years old, with a high incidence rate in women before and after menopause. Breast cancer is mainly caused by heredity, eating disorders and radiation, the incidence rate of which has been increasing with the development of electronic industry and changes of living standards. The disease is clinically manifested as breast mass, axillary lymph node enlargement and breast pain, the mortality rate of which is high due to its unapparent symptoms and patients’ little attention to the condition –21.Early diagnosis and treatment are important for the prognosis of breast cancer, and miRNA can be used as a diagnostic marker because it is specifically expressed in the diseased tissue and serum. miRNA is specific in the tissue and plasma of patients with tumors, indicating that miRNA can be used as a new tumor marker . AccordiP <0.05). Moreover, miR-21 and miR-210 were gradually up-regulated with the severity of the disease. It is suggested that miR-21 and miR-210 have diagnostic value in breast cancer, similar to another study , whereas there was no significant difference between the miR-21 high and low expression groups (P > 0.05), indicating that miR-210 can be used as a biomarker for predicting the prognosis of breast cancer patients. Different miRNAs have different expressions in the same tumor, so further research is needed on this direction. High expression of miR-210 was an independent factor, and low expression of miR-210 showed better disease-free survival and overall survival than high expression (P <0.05)  , which iIn recent years, miR-21 and miR-210 are hot topics in clinical research. The expression and prognostic values of them in patients with breast cancer were comprehensively explored in this paper, which is hoped to provide references for clinical research. However, there are still some limitations. The sample size of this paper is small, the upstream and downstream regulatory genes and specific mechanisms of miR-21 and miR-210 have not been studied. In the future research, the prediction of treatment outcomes by different distribution on molecular subtypes of the two RNAs can be further explored, in order to help more correctly judge the prognosis.The study of miR-21 and miR-210 expressions is helpful to know the occurrence, development and biological behavior of breast cancer. miR-21 and miR-210 expressions are significantly correlated with lymph node metastasis, clinical staging and differentiation, and miR-210 can be used as a biomarker for the prognosis of breast cancer.Ethical issues  have been completely observed by the authors.


There is growing interest in developing biomaterial-coated liposome delivery systems to improve the stability and bioavailability of curcumin, which is a hydrophobic nutraceutical claimed to have several health benefits. The curcumin-loaded rhamnolipid liposomes (Cur-RL-Lips) were fabricated from rhamnolipid and phospholipids, and then chitosan (CS) covered the surface of Cur-RL-Lips by electrostatic interaction to form CS-coated Cur-RL-Lips. The influence of CS concentration on the physical stability and digestion of the liposomes was investigated. The CS-coated Cur-RL-Lips with RL:CS = 1:1 have a relatively small size (412.9 nm) and positive charge (19.7 mV). The CS-coated Cur-RL-Lips remained stable from pH 2 to 5 at room temperature and can effectively slow the degradation of curcumin at 80 °C; however, they were highly unstable to salt addition. In addition, compared with Cur-RL-Lips, the bioavailability of curcumin in CS-coated Cur-RL-Lips was relatively high due to its high transformation in gastrointestinal tract. These results may facilitate the design of a more efficacious liposomal delivery system that enhances the stability and bioavailability of curcumin in nutraceutical-loaded functional foods and beverages. Liposomes (Lips) are spherically shaped vesicles consisting of a phospholipid bilayer that surrounds an aqueous core; they have attracted widespread attention for their good nutraceutical-loaded property . Due to d-glucosamine and N-acetyl glucosamine units linked through a β (1 → 4) linkage [3 function on repeating the d-glucosamine unit [Chitosan (CS) is obtained by partial deacetylation of chitin from crustacean shells, which consists of  linkage . When CSine unit . Based o a β 1 →  linkage ine unit . Furtherine unit . Take thIn this study, curcumin (Cur) was utilized as a model bioactive substance due to its good activity, such as antioxidant, anticancer, antibacterial, and anti-inflammatory . The useThe formation of CS-coated Cur-RL-Lips was mainly attributed to the electrostatic interaction between CS with positive charge and Cur-RL-Lips with negative charge, as schematized in Additionally, the microstructure of the liposomes was obtained using AFM topographic images . The CurThe liposomes could experience various environmental stresses when they were added to commercial products  or when they travel through the human gastrointestinal tract (GIT) ,25. ThesLiposome suspensions were exposed to various pH values (pH 2~7) for 3 days, and then their visual appearance, particle size, and surface charge were measured. As shown in The salt stability of the liposomes was determined by adding different levels of NaCl (0~400 mM) to them . The turP < 0.05) in the particle size of liposomes both before and after storage  is important for their commercial applications. Hence, the thermal stability of Cur-loaded liposomes was determined by measuring the curcumin degradation and the change of particle size at 80 °C for 60 min . The con storage b. TherefThe in vitro release behavior of curcumin was recorded during 72 h at 37 °C, and the cumulative release curve is shown in The digestion process of the liposomes was studied by a three-stage simulated GIT model, including mouth stage, stomach stage, and small intestine stage. At the end of each single stage, the visual appearance, particle size, and ζ-potential were recorded a–c. WheninitialC), as well as the curcumin concentration in raw digesta phase (digestaC) and in mixed micelle fraction (micelleC) at the end of small intestine stage. Then, these data were used to calculate the transformation (T*) and bioaccessibility (B*), as the following equations:Additionally, the in vitro bioavailability of curcumin was determined by measuring initial curcumin concentration (The bioavailability (BA) is given by B* × T*, so it is a ratio of the curcumin concentration in mixed micelle fraction to the initial curcumin concentration. As shown in Phospholipid S100 (containing 94% phosphatidylcholine) was purchased from Lipoid GmbH . Curcumin (98%) was purchased from Aladdin Industrial Corporation . Chitosan was purchased from Sigma-Aldrich Co. . Rhamnolipids were provided by Xi’an boliante Chemical Co., . All other reagents  were purchased from Xilong Chemical Co., . Double distilled water (Milli-Q) was used to prepare all solutions.The curcumin-loaded rhamnolipid liposomes (Cur-RL-Lips) were prepared according to the method of Cheng et al.  with somw/w). Finally, the Cur-RL-Lips (control) and CS-coated Cur-RL-Lips with different RL:CS mass ratios were adjusted to pH 4.5 by adding a small amount of either HCl or NaOH solution.The Cur-RL-Lips were coated with chitosan using the method of Cuomo et al.  with somThe mean diameter and ζ-potential of the liposomes were determined using an instrument capable of both dynamic light scattering and microelectrophoresis analysis  according to the method of Peng et al. . The lipThe microstructure of liposomes was observed using an atomic force microscope (AFM). The liposome suspensions were dropped into the clear mica plate, and then the microstructure images of liposomes were obtained by an AFM  with a silicon cantilever of force constant of 0.58 N/m operated in tapping mode at room temperature.pH: The influence of pH on the stability of liposome dispersions was determined by preparing a series of samples with the desired pH values ranging from 2 to 7. The obtained samples were then stored in a 25 °C incubator for around 24 h and 3 d, respectively. The appearance, mean particle diameter, and ζ-potential of the samples were recorded using the methods described earlier.Salt addition: The influence of salt level on the stability of liposome suspensions was determined by preparing a series of samples with different levels of NaCl . The obtained samples were then stored in a 25 °C incubator for around 24 h and 3 d, respectively. The appearance, mean particle diameter, and ζ-potential of the samples were recorded using the methods described earlier.Thermal stability: The liposome suspensions (5 mL) were mixed with 20 mL of phosphate buffer solution  and sealed into a series of 2 mL test tubes, which were incubated in a water bath at 80 °C for 10, 20, 30, 40, 50, and 60 min, respectively. The tubes were then immediately cooled to room temperature using an ice water. Finally, the mean particle diameter was determined using the methods described earlier and the curcumin concentration was obtained by measuring the absorbance of samples at 420 nm.w/w) and 10% ethanol as an auxiliary solvent for curcumin and incubated in a shaker at 100 rpm and at 37 °C for 72 h. At specific time intervals, 2 mL of release medium was withdrawn and replaced with fresh medium. The release medium was diluted with anhydrous ethanol to calculate the total cumulative amount of curcumin released from the liposomes. The content of curcumin was measured at various sampling times by a spectrophotometer at 420 nm.The in vitro release of curcumin from liposomes was evaluated according to the method of Li et al. . Briefly0V is the initial volume of curcumin in release system, 0M is the initial mass of curcumin in the sample, and iC is the concentration of curcumin released at each sampling time point.The cumulative release (%) of curcumin was calculated using the equation below:A three-stage simulated GIT model was employed to characterize the potential gastrointestinal behavior of the liposomes ,36. (i) p < 0.05 by Tukey’s HSD (honestly significant difference) test.All experiments were performed on three prepared samples. The results were expressed as mean values ± standard deviation in triplicates. Statistical analysis software (SPSS 25.0) was used for analyzing the data. Analysis of variance (ANOVA) was used to determine significance at In this study, the impact of the ratios of chitosan (CS) and rhamnolipid (RL) on the physical stability and digestion of the chitosan-coated rhamnolipid liposomes containing curcumin (CS-coated Cur-RL-Lips) was examined. Initially, we showed that chitosan (CS) and Cur-RL-Lips spontaneously assembled into CS-coated Cur-RL-Lips in aqueous solutions because of electrostatics interaction between cationic CS and anionic Cur-RL-Lips. Then, the physical stability and gastrointestinal digestion of CS-coated Cur-RL-Lips were investigated. The CS-coated Cur-RL-Lips were stable to aggregation under acidic conditions (pH ≤ 5), which is conducive to enduring the acidic condition of stomach and preventing the release of the encapsulated drug in the stomach. The CS-coated Cur-RL-Lips exhibited better stability than the Cur-RL-Lips when stored at elevated temperatures (80 °C for 60 min). The CS covered the surface of liposomes effectively inhibited temperature-induced curcumin degradation. Additionally, curcumin in CS-coated Cur-RL-Lips can be better absorbed than those in Cur-RL-Lips, but a high level of CS reduced the bioaccessibility of curcumin. Therefore, the amount of CS in liposomes should be controlled reasonably. Overall, these results have good potential for the design of a more efficacious liposomal delivery system for encapsulating and delivering bioactive agents.


Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease, which is a chronic granulomatous enteropathy in ruminants. Determining the genetic diversity of MAP is necessary to understand the epidemiology and biology of MAP, as well as establishing disease control strategies. In the present study, whole genome-based alignment and comparative analysis were performed using 40 publicly available MAP genomes, including newly sequenced Korean isolates. First, whole genome-based alignment was employed to identify new genomic structures in MAP genomes. Second, the genomic diversity of the MAP population was described by pangenome analysis. A phylogenetic tree based on the core genome and pangenome showed that the MAP was differentiated into two major types (C- and S-type), which was in keeping with the findings of previous studies. However, B-type strains were discriminated from C-type strains. Finally, functional analysis of the pangenome was performed using three virulence factor databases  to predict the phenotypic diversity of MAP in terms of pathogenicity. Based on the results of the pangenome analysis, we developed a real-time PCR technique to distinguish among S-, B- and C-type strains. In conclusion, the results of our study suggest that the phenotypic differences between MAP strains can be explained by their genetic polymorphisms. These results may help to elucidate the diversity of MAP, extending from genomic features to phenotypic traits. Mycobacterium avium subsp. paratuberculosis (MAP) is a causative agent of Johne’s disease, which is a chronic granulomatous enteropathy in ruminants. MAP infection is characterized by chronic diarrhea, progressive wasting, and eventual death. MAP infection is also economically important because infected individuals exhibit weight loss and reduced milk production [oduction . The disoduction . In addioduction .1311 [Determining the genetic diversity of MAP is necessary to understand the epidemiology and biology of MAP, as well as establishing disease control strategies , 5. MAP 1311 . Some mo1311 , 9. More1311 , 10. Des1311 –15.The increased accuracy and lower cost of WGS has facilitated efforts to accumulate WGS data, which enabled researchers to perform comparative genomic analyses. Comparative genomics is a widely employed analytical method to identify differences or similarities among bacteria . Among tAlthough there are many studies based on the whole genome of MAP, more whole genome data of various strains of MAP worldwide are still needed. In particular, there is no complete level of genome sequences for strains known as B-type. Therefore, in this study, complete genome sequencing was performed on B-type strains, which account for a large proportion of the strains detected in Korea, and C-type strains isolated in the same region. Subsequently, the standard strain of MAP and the newly sequenced genome derived from Korea were compared and analyzed using the genome alignment technique. New large gaps and novel tandem repeats (TRs) were observed from the whole genome alignment between the Korean strains and reference genome K-10. Furthermore, pangenome analysis was performed using the whole genome database of MAP to describe the genomic features of the MAP with respect to genetic diversity, especially in virulence-related genes. Through the analysis, we also attempted to define the links between population structure and pathogenicity. Pangenome analysis was performed with 40 MAP genomes and confirmed that MAP has a well-conserved genome, even though there were a large number of S-type-specific genes. The virulence factors (VFs) were searched in the pangenome with three different databases. It was confirmed that most of the known VFs were conserved in all genomes. Finally, we designed a real-time PCR technique with a novel biomarker gene identified in this study to efficiently distinguish the type of MAP strains.1311 PCR-REA, MIRU-VNTR, and MLSSR typing was employed to differentiate the isolates into 5 different groups. In this study, five isolates were selected for WGS based on genotyping results: three B-type isolates  and two C-type isolates   following the manufacturer’s instructions (Pacific Biosciences). The constructed library was validated by an Agilent 2100 Bioanalyzer. The SMRTbell library was sequenced using SMRT cells (Pacific Biosciences) using C4 chemistry (DNA sequencing Reagent 4.0), and 240 min movies were captured for each SMRT cell using the PacBio RS II (Pacific Biosciences) sequencing platform . De novoWhole-genome sequence data of five newly sequenced MAP strains and 35 other MAP strains were downloaded from NCBI, and reannotation was performed by using RAST . Even thWhole-genome sequence alignment of 14 MAP genomes was performed by the Mauve alignment tool (ver. 20150226) with default options of the progressiveMauve algorithm. Except for the JIII-386 strain, all MAP genomes were complete genomes. New large gaps and tandem repeats were discovered from this analysis, and the found information was confirmed by utilizing the NCBI database.N is the total number of isolates in the typing scheme, s is the total number of distinct types discriminated by each typing method, and nj is the number of isolates belonging to the jth type.Novel tandem repeats (TRs) identified in this study were further analyzed with 13 complete genome sequences of MAP available in NCBI. The genotype of each genome was verified by aligning the sequences of the TR region (TR type). The discriminatory index (DI) described by Hunter and Gaston  was usedThe Bacterial Pangenome Analysis (BPGA) Tool  was usedFunctional analysis was performed based on the Basic Local Alignment Search Tool (BLAST) algorithm. USEARCH  was usedn = 9), Czech (n = 1), and M. avium subsp. hominissuis strain 104 and 101 (n = 2). Each isolate was cultured in 5 mL of Middlebrook 7H9 broth supplemented with Middlebrook OADC and mycobactin J (2 mg/L). After incubation at 37 °C for 6 weeks, the bacterial suspension was centrifuged at 3000 × g for 20 min, and the pellet was lysed in 1 mL of L6 lysis buffer . Subsequent processes were performed as previously described [A total of 12 isolates were obtained from Korea (escribed . All PCRM. avium subsp. paratuberculosis (MAP) strains that were isolated from Korea as previously described [Genome sequencing was conducted with five escribed . Three s256 family transposase (MAP_RS19260) and TetR/AcrR family transcriptional regulator (MAP_RS22345), which were located at approximately 4.2 Mb and 11 kb in K-10. This inverted sequence was also found in other MAP genomes. Two strains (JII-1961 and TANUVAS) showed the same form as K-10, while other C- and B-type MAPs showed inverted forms  and generated two additional transposases, IS481-like element ISMav5 family transposase  and IS256 family transposase . A notable feature of this gap was that its inserted location was the end of the genome inverted region . This discovery was confirmed by determining that three strains  have 17 conserved regions that have nucleotide sequences similar to those of IS900, while the other three strains  have 16 conserved regions. The gap was inserted into the middle of acyl-ACP desaturase , separating it into two pseudogenes .The last large gap was a 1452 bp gap, which could be observed in K-10, MAPK_JB16, and MAPK_JJ1 Figure A. The nu1311 PCR-REA. The discriminatory index (DI) with the novel TRs was calculated as 0.795 among the 13 genomes, whereas the DI value of the original MIRU-VNTR was 0.628. When the eight existing TRs and five novel TRs were considered together, the DI value was calculated as 0.872.Among the smaller gaps found in this study, seven sequences were found to be TRs Table . Two of Genomic comparison was performed using the 40 MAP whole genome sequences obtained from NCBI  database . Through1311 element [Pangenome-based phylogenetic analysis was performed among 40 MAP strains for the identification of genetic diversity, especially the classification of strains. Major genotypes of MAP have been employed to classify the strains into three types, S-, B-, and C-type, which are distinguished by polymorphisms of the IS element .Figure 6In the case of the panphylogeny Figure B, a treembt, mce, and mmpL genes.From the results of pangenome analysis, VF-related genes were searched with three different databases, that is, VFDB, Victors, and PATRIC –27. AmonmbtA, but the former was a gene found only in S-type genomes, while the latter was a gene found in the rest of the genomes. It was reported that the S-type genome has different lengths and sequences in the mbtA and mbtE genes [There were 13 type-specific genes that were related to VFs  were identified as mbtH, two clusters (62 and 206) were identified as mbtB, two clusters (88 and 1991) were identified as mbtD, two clusters (671 and 1010) were identified as mbtI, and two clusters (3582 and 4183) were identified as mbtK. Other mbt genes, mbtC, mbtG, and mbtJ, were found to match clusters 1075, 1131, and 2318, respectively.The  to mbtJ . Twenty-mceA to mceF [mce gene clusters [mce-family proteins was 59 in the pangenome  was also returned as a hit to the mce gene from the BLASTp results. Three interesting results were obtained through this comparison. First, absence of one mce cluster was observed. It was found that the JIII-386 genome had seven mce clusters, while the other genomes had eight [mce2 cluster that had only mceA and mceB genes in the cluster region. Second, type-specific point mutations in certain mce genes were identified. Cluster 1862, which is identified as mce4B, had two point mutation positions depending on type. The S-type, B-type, and four C-type  have mutations (Cys → Arg) in the 62nd amino acid sequence. Additionally, S-type genomes had additional mutations  on the 245th amino acid sequence of the same cluster. Cluster 1955, which was identified as mce7B, also had an S-type specific mutation (Lue → Phe) on the 282nd amino acid sequence. Third, CDSs that compose the cluster 850, identified as mce7D, consisted of one long CDS in the S-type genome and two sequential short CDSs in other genomes.The mce genes are cell-wall proteins that generally form a cluster consisting of five genes from  to mceF –38. It wclusters . The numad eight . The JIImmpL) proteins are essential genes for physiological and pathogenetic reasons [mmpL in the pangenome, and eight mmpL genes were returned as hits from the BLASTp results  into one group and consider them core genes with mmpL6 because the protein sequences were very similar. Three clusters  should also be considered as one cluster with the mmpL3 gene, which is well conserved in all MAP genomes.The mycobacterial membrane protein large  when searching the accessory genome. The predicted function of the three clusters was the same as that of the MoaD/ThiS family protein -based detection method was designed. The genome region 92 bp upstream of the gene was selected as the forward primer binding region, and the reverse primer binding region was selected on the repeated region (repeat of GTG) for differential amplification depending on the type Figure C. Real-tM. avium strains, M. avium subsp. avium 101 (MAA 101) and M. avium subsp. hominisuis 104 (MAH 104), have the same gene as the S-type strain; therefore, two M. avium strains were used for PCR verification.The expected result was that the S-type strain was specifically amplified only in primer RS, whereas the C-type strain was specifically amplified for primers RS and RC, and the B-type was specifically amplified in all primer sets Figure . Five stReal-time PCR was successfully performed, and it appears that novel primers can distinguish the type of MAP  is an important pathogen in the dairy cattle industry due to its economic impact [c impact , and it c impact . It is ic impact , which hc impact –45. Compc impact , 39, 46.M. avium subsp. hominissuis (MAH) strains, including strains 104 and TH135, with 100% query coverage and 98.6% and 98.45% identity from the NCBI BLAST results (data not shown). Based on the CDSs in the MAH 104 strain, two proteins, acyltransferase domain-containing protein (MAV_RS14825) and type I polyketide synthase (MAV_RS14830), were annotated in the region covering the 6204-bp sequence. BLAST results of the MAPK_JJ1 and MAH 104 strains showed deletion of two consecutive nucleotide sequences in MAPK_JJ1. This finding might have resulted in the annotation of pseudogenes in MAPK strains. Taken together, these results suggest that a 6204-bp sequence was deleted in strains K-10 and MAPK_CN9.In this study, we performed whole genome sequence (WGS) alignment to determine the genetic diversity of the MAP strains. It was found that there is a chromosomal change in the MAP genomes. All C-type and B-type genomes had partially inverted genomes compared to reference K-10, except TANUVAS and JII-1961. The inversion of the genome could be used as a new criterion to distinguish MAP strains, since they have highly conserved genomes. Another interesting point of genome inversion was that there was a large gap in Korean isolates in the position of the inversion. This gap was one of the large gaps we observed, measuring 2026 bp in size, and it contained a transposase. It was reported that both phenomena, genome inversion and transposition, often result in gene duplication , 48. ThuThe genotyping of MAP strains was classified by using the INMV database , which dPangenome analysis was performed to compare the MAP genomes after WGS alignment. Pangenome analysis is a useful tool for comparing genome data to identify the common features and differences of genomes at the same time. Through the analysis, it was confirmed that MAPs have well-conserved genomes and that their pangenomes are closed pangenomes. However, some clusters were formed incompletely. Even though the genome data were filtered before pangenome analysis, 15 of 40 genomes had low quality and had more than 450 contigs , which is a notably small number. Even though the number of tested samples was small, PCR experiments were performed under various conditions. Additionally, from this experiment, we confirmed that real-time PCR could be employed to check single nucleotide polymorphisms (SNPs) in the genes. Furthermore, in the same gene, the repeat count of A on the 23Additional file 1.Circular visualization of genomic reconstruction for the five MAPK strains. (A) MAPK_CN4/13. (B) MAPK_CN7/15. (C) MAPK_CN9/15. (D) MAPK_JB16/15. (E) MAPK_JJ1/13.Additional file 2.Whole genome sequence alignment of 14 complete genomes available in public databases. Sequence alignment with Mauve software showed that two strains (JII-1961 and TANUVAS) had same form with K-10, while other C- and B-type MAPs had inverted forms.Additional file 3.Genomic gaps between MAPK strains and the K-10 strain identified by Mauve analysis.Additional file 4.List of whole genome sequences analyzed in this study.Additional file 5.List of pangenome data.Additional file 6.Pangenome and core genome plots analyzed by the Bacterial Pangenome Analysis tool. Pan and core genome plot showed that MAP genome has almost closed genome.Additional file 7.Cluster of genes identified as S-, B-, C-type specific.Additional file 8.Phylogenetic relationship between 40 MAP strains based on core genome phylogeny with amino acid sequences. Amino acid sequence-based core genome phylogeny showed similar result with nucleotide-based core genome phylogenetic analysis.Additional file 9.List of genes mapped to virulence factor databases .Additional file 10.Amplification plot of newly designed type-discriminating real-time PCR using 12 different MAP strains. Representatives of S-type (M. avium strain 101 and 104) showed only single specific amplification, whereas C-type strains showed double specific amplification. B-type strains were amplified with all three primers.